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Patent Issued for Method for the Intensive Extraction of Cellular Compounds from Micro-Organisms by Continuous Culture and Extraction, and...

May 20, 2014

Patent Issued for Method for the Intensive Extraction of Cellular Compounds from Micro-Organisms by Continuous Culture and Extraction, and Corresponding Device

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent by the inventors Pruvost, Jeremy (Saint Brevin, FR); Legrand, Jack (Saint Nazaire, FR); Foucault, Alain (Saint Nazaire, FR), filed on April 22, 2008, was published online on May 6, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Universite de Nantes).

Patent number 8715956 is assigned to Universite de Nantes (Nantes Cedex, FR).

The following quote was obtained by the news editors from the background information supplied by the inventors: "One of the benefits of photosynthetic micro-organisms such as microalgae and cyanobacteria lies in the original composition thereof.

"However, the majority of these compounds involve the application of stress conditions to the culture so as to make use of the significant metabolic flexibility of this type of micro-organism to achieve the biosynthesis of the desired compound.

"If this compound is intracellular (such as pigments in particular), it is necessary to implement a second extraction step which, in the majority of cases, causes irreversible damage to the culture (grinding, thermal or osmotic shock, cellular disintegration, etc.).

"Therefore, industrial productions based on this principle use discontinuous productions, successively alternating biomass production, stress condition application phases, followed by harvesting, extraction and purification phases.

"The main drawback of discontinuous productions is associated with the low growth rate of photosynthetic micro-organisms (compared to heterotrophic micro-organisms such as bacteria or yeasts), preventing frequent harvesting, or, at the very least, involving work with a plurality of production systems in parallel.

"For some compounds, it is possible to implement an original extraction technique, wherein only the compound is extracted, without altering the cell significantly (biocompatible extraction). This enables the continuous production of the compound, while preventing the repetition of the growth and stress phase.

"Indeed, if this technique is associated with continuous biomass production in a photobioreactor, once the initial growth phase has been carried out, the stress conditions subsequently applied can be theoretically maintained indefinitely, provided that the compound produced is continually extracted.

"This results in a non-negligible gain in production compared to discontinuous methods, avoiding the losses associated with the latent period before obtaining a further biomass having the desired cellular composition.

"Such a continuous production-extraction technique was proposed by Hejazi and Wijffels in 2003 (patent document published under the number EP-1 501 937) and is described with reference to FIG. 1.

"This device is also based on the research detecting the existence of biocompatible solvents enabling the extraction of reusable compounds, in this instance .beta.-carotene from the microalga Dunaliella salina (research by Leon et al., 2003).

"As illustrated in FIG. 1, this device comprises a photobioreactor wherein the culture 1 containing the compound to be extracted and the solvent 2 wherein the compound is progressively extracted coexist (biphase system).

"Although it is operational, this method displays various limitations. Indeed, the extraction rate remains relatively low, due to the difficulty placing the two non-miscible phases (cultures in aqueous medium and hydrophobic solvent) in contact. As the contact interface remains small, there is a significant resulting limitation in material transfer. The solution consists of mixing the whole very vigorously, which, however, rapidly reaches a limit determined by the fragility of the cultured cells.

"A further drawback lies in the impossibility to impose optimal conditions, as both processes (biosynthesis and extraction) are different. For example, as extracted .beta.-carotene is photosensitive, the use of strong lighting (stress condition required for biosynthesis) for culture makes it necessary to draw off the solvent charged with pigment regularly before degradation."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "An aspect of the disclosure relates to a method for the extraction of cellular compounds from micro-organisms, of the type that comprises: a culture step of said micro-organisms; an extraction step of said cellular compounds from said micro-organisms,

"characterised in that each step is carried out in a continuous manner, said extraction step being carried out separately from said culture step, and said extraction step being carried out under conditions of biocompatibility with said micro-organisms, followed by: at least one step for recovering said cellular compounds; at least one step for recirculating said micro-organisms towards said culture step.

"In that the method is based on two separate and continuous cellular compound culture and extraction steps, an embodiment of the invention is applicable both to photosynthetic micro-organisms and to non-photosynthetic micro-organisms (bacteria, yeasts). Only the culture step differs, the photobioreactor (in the case of photosynthetic micro-organisms) possibly being replaced by any other bioreactor suitable for the micro-organism used.

"Since each of the phases (production by means of culture and extraction) is based on very different principles, technologies and parameters, the operation in two subsystems according to an embodiment of the invention makes it possible to obtain an intensified overall method with the option of enhanced and independent control of each subsystem.

"It should be noted that an embodiment of the invention is particularly applicable to the extraction of an intracellular compound. However, in some cases, the micro-organisms may release a metabolite (extracellular metabolite) in the medium. An embodiment of the invention is also applicable to this case, with the same overall operating principle, the only difference being that the metabolite is then extracted from the culture medium by the extractor (liquid-liquid extraction), instead of directly from the cell (solid-liquid extraction).

"However, whether the metabolite is intra- or extra-cellular, the looped operating concept of both subsystems is only possible if the extraction phase is not destructive with respect to the biological material, so that the living cells are reintroduced into the production subsystem.

"Otherwise, both subsystems operate in cascade, which is a conventional industrial format. Therefore, the extraction step should prevent irreversible damage to the culture, whether from mechanical, thermal or chemical causes. A suitable choice of the extraction system makes it possible to prevent mechanical damage and thermal shocks.

"Therefore, the originality of an embodiment of the invention lies in the division into two optimised subsystems, operating in a coupled manner, each complying with the constraints eventually enabling continuous production-extraction. Compared to the existing system, two major benefits are provided: option to increase material transfers at the extractor (and therefore improved overall yield); option to apply different conditions for extraction than for production to preserve the integrity of the intracellular compound extracted.

"In sum, an embodiment of the invention enables major improvements both with respect to material transfer and with respect to the option offered to apply optimal operating conditions for both steps (production and extraction), by implementing two subsystems dedicated for each step and operating in a coupled manner, i.e. with continuous production and under intracellular compound stress conditions, and a biocompatible extraction part with respect to said compound.

"It should be noted that, even if, in principle, the complexity of the method is increased by the use of the two subsystems, the coupling is facilitated by the operation in continuous and permanent mode. It is thus possible to optimise the culture conditions to improve extraction (maintaining a constant intracellular concentration over time enables better definition of the extraction conditions to be applied).

"According to one advantageous solution, said culture step is a bioproduction step, providing the live medium with the conditions required for the synthesis of the desired metabolite.

"Advantageously, said extraction step is a liquid-liquid (extracellular metabolite) or solid-liquid (intracellular metabolite) extraction step.

"Such an extraction mode ensures the transfer of the desired metabolite from the cell (case of an intracellular compound) and/or the culture medium (case of an exuded extracellular compound) to a solvent subsequently recovered at the extractor outlet.

"According to a preferred solution, said extraction step is performed by means of Centrifugal Partition Chromatography (CPC).

"In this way, the high transfer capacities of CPC make it possible to obtain a high-performance liquid-liquid extraction system.

"However, the use of CPC involves a major constraint, i.e. the possibility of high mechanical stress subjected to the live cells while passing through the apparatus (centrifugal field, culture pumping system, shearing between and in each separation cell). Therefore, the system is only suitable for continuous use if the culture is capable of withstanding this treatment. Tests have demonstrated the feasibility of the approach and the potential thereof (increased yields with respect to the literature), subject, however, to remaining within an acceptable CPC operating range to preserve the integrity of the biological material.

"According to one possible alternative embodiment, said extraction step is carried out protected from light.

"According to a further possible alternative embodiment, said extraction step is carried out in anoxia in gaseous nitrogen.

"Therefore, it is understood that a method according to an embodiment of the invention enables the ready application of the specific conditions in the extraction system, such as working protected from light if the product is photosensitive, or under anoxia conditions, if the product is oxidised rapidly. Similarly, the extraction mode adopted may be selected to achieve a high selectivity (choice of solvent and extraction time control).

"An embodiment of the invention also relates to a device for the implementation of a method as described above, comprising: at least one vessel for the culture of said micro-organisms, at least one vessel for the extraction of said cellular compounds from said micro-organisms, said extraction vessel being separate from said culture vessel and containing at least one solvent biocompatible with said micro-organisms; means for recovering said cellular compounds; means for recirculating said micro-organisms from said extraction tank in said culture vessel.

"It is noted that an embodiment of the invention prevents chemical damage to the micro-organisms, by using a biocompatible solvent, which extracts the intracellular metabolite, without altering the metabolism of the micro-organism irreversibly potentially leading to cellular lysis.

"According to one advantageous solution, said culture vessel is a photobioreactor, and said extraction vessel is a liquid-liquid extraction vessel."

URL and more information on this patent, see: Pruvost, Jeremy; Legrand, Jack; Foucault, Alain. Method for the Intensive Extraction of Cellular Compounds from Micro-Organisms by Continuous Culture and Extraction, and Corresponding Device. U.S. Patent Number 8715956, filed April 22, 2008, and published online on May 6, 2014. Patent URL:

Keywords for this news article include: Universite de Nantes.

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Source: Life Science Weekly

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